EP2118306B1 - Verfahren zum nachweis methicillinresistenter s. aureus sowie primer, sonden und kits dafür - Google Patents

Verfahren zum nachweis methicillinresistenter s. aureus sowie primer, sonden und kits dafür Download PDF

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EP2118306B1
EP2118306B1 EP07857150A EP07857150A EP2118306B1 EP 2118306 B1 EP2118306 B1 EP 2118306B1 EP 07857150 A EP07857150 A EP 07857150A EP 07857150 A EP07857150 A EP 07857150A EP 2118306 B1 EP2118306 B1 EP 2118306B1
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mrsa
probe
mec
sample
seq
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EP2118306A1 (de
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Christian Aichinger
Astrid Reiser
James R. Uhl
Franklin R. Cockerill
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
Mayo Foundation for Medical Education and Research
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
Mayo Foundation for Medical Education and Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to methods of detecting the presence or absence of methicillin-resistant S. aureus (MRSA) in a sample, the method comprising performing an amplifying step, a hybridizing step and a detecting step. Furthermore, the present invention relates to primers, probes and kits for the detection of MRSA.
  • MRSA methicillin-resistant S. aureus
  • Staphylococcus aureus ( S. aureus; SA) is a bacterium, whose natural reservoir is mucous or wet/salty skin areas like the groin, anus or nose. In addition it inhabits wounds. Overall, the nose is the predominant environment for S. aureus. However, S. aureus can cause illnesses ranging from minor skin infections (such as pimples, boils, and cellulites) and abscesses, to severe diseases such as pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), septicemia and Multi Organ Dysfunction Syndrome (MODS). Each year some 500,000 patients in American hospitals contract a staphylococcal infection.
  • MRSA may also be known as oxacillin-resistant Staphylococcus aureus (ORSA) and multiple-resistant Staphylococcus aureus, while non-methicillin resistant strains of S. aureus are sometimes called methicillin-susceptible Staphylococcus aureus (MSSA) if an explicit distinction must be made.
  • ORSA oxacillin-resistant Staphylococcus aureus
  • MSSA methicillin-susceptible Staphylococcus aureus
  • MRSA Newcastle disease virus
  • unrecognized colonization with MRSA may lead to the distribution of MRSA from one patient to the other by replacing the flora of MRSA-uncolonized patients with MRSA. Colonization is eased by certain risk factors including e.g. previous antibiotic treatment, polymorbidity, and diabetes as well as previous stays in hospital.
  • the diagnosis of most skin infections is made by the pattern of symptoms and physical exam findings but it is not usually possible to know whether the infection is caused by Staphylococcus bacteria of any type or another bacterium, like e.g. group A Beta-hemolytic Streptococcus ( Streptococcus pyogenes ).
  • Staphylococcus bacteria of any type or another bacterium like e.g. group A Beta-hemolytic Streptococcus ( Streptococcus pyogenes ).
  • a culture can be done.
  • diagnosing MRSA using a conventional culture requires 16-72 hours, causing a delay in treatment, significantly impairs patient outcomes and may facilitate outbreaks as well as increase hospital costs.
  • Phenotypic analyses such as coagulase type, enterotoxin (SE) type, production of toxic shock syndrome toxin-1 and in vitro antibiotic susceptibility, have been used routinely for MRSA strain typing.
  • genotypic analysis for MRSA strains has been established, providing a more detailed classification than phenotypic analysis.
  • pulse-field gel electrophoresis (PFGE) using restriction fragments of total genomic DNA of MRSA is an excellent method of characterization.
  • multilocus sequence typing may be used in order to characterize isolates of bacteria by using the sequences of internal fragments of seven housekeeping genes.
  • MLST has been developed and validated for S. aureus ( Enright et al. 2002, Proc. Nat. Acad. Sci. U.S.A. 99: 7687-7692 ) and provides a discriminatory method that allows related strains recovered in different countries to be readily identified.
  • EP 1529847 discloses a method for detecting MRSA using primers specific for the night junction of SSC mec.
  • Test kits have been developed in order to detect MRSA (e. g. IDI-MRSA assay, Becton Dickinson, USA) which is an in vitro diagnostic test for the direct detection of nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) to help prevent and control MRSA infections in healthcare settings.
  • MRSA methicillin-resistant Staphylococcus aureus
  • this kit has a series of disadvantages which limit its usefulness.
  • SCC Staphylococcal Chromosomal Cassette
  • the inhibition rate is quite high and, finally, a numerousness of primers and probes used is required, resulting in a diagnostic tool, which is prone to errors.
  • melting curve analysis which may be used by the FRET probe assay allows a higher specificity within the output signal compared to amplification curves. Therefore, one object of the present invention was to provide an alternative method for the detecting of MRSA, preferably an alternative method avoiding the above disadvantages.
  • the object of the present invention has been solved by providing a method of detecting the presence or absence of methicillin-resistant S . aureus (MRSA) in a sample, the method comprising
  • the method may be used for fast and reliable detection of MRSA in a sample. Particularly, the method may be used in order to detect MRSA in a sample or diagnose an MRSA infection of an individual, such as a patient.
  • a limited number of primers may be used in order to detect members of all presently known SCC mec types, i.e. types I, II, III, IV and V which provides to a highly reliable tool for the detection of MRSA.
  • the method of the present invention is suitable for detection MRSA of all known Staphylococcal Chromosomal Cassettes (SCC mec ) types, i.e. type I, II, III, IV and V. This is particularly important in order to choose the appropriate treatment e.g. for a patient infected with MRSA.
  • SCC mec Staphylococcal Chromosomal Cassettes
  • MecA The methicillin resistance gene ( mecA ).
  • MecA encodes an altered methicillin-resistant penicillin-binding protein (PBP2a or PBP2'), a penicillin binding protein with reduced affinity for ⁇ -lactam rings (the primary active-site of the ⁇ -lactam antibiotics such as penicillins, cephalosporins and carbapenems) ( Guignard B et al., 2005, Curr Opin Pharmacol 5 (5): 479-89 ), that is not present in susceptible strains and is believed to have been acquired from a distantly related species.
  • PBP2a or PBP2' penicillin binding protein with reduced affinity for ⁇ -lactam rings
  • ⁇ -lactam antibiotics such as penicillins, cephalosporins and carbapenems
  • MecA is carried on a mobile genetic element, the Staphylococcal Chromosomal Cassette mec (SCC mec ) of MRSA strains, of which five forms have been described that differ in size and genetic composition. SCC elements also occur in sensitive S . aureus but do not carry the mecA gene or carry a non functional mecA gene. Such strains are a major source of false positive results, because they do have the same right extremity junction.
  • SCC mec Staphylococcal Chromosomal Cassette mec
  • MRSA detection from nasal specimen by detecting the mecA gene and a S. aureus specific gene leads to low positive predictive values (PPV) due to the presence of varying amounts of both non resistant SA and methicillin-resistant coagulase-negative staphylococci (MRCoNS).
  • PPV positive predictive values
  • MRCoNS methicillin-resistant coagulase-negative staphylococci
  • a combination of those is undistinguishable from MRSA, because of the presence of both targets.
  • MRCoNS methicillin-resistant coagulase-negative staphylococci
  • SCC mec Staphylococcal Chromosomal Cassette
  • SCCmec the SCC element of MRSA (with functional mec A gene), is a transposon of a length of 16 kb - 67 kb integrated into the 3' portion of the open reading frame X from S. aureus (orfX) containing the mecA gene.
  • MecA encodes the PBP2a conferring resistance to methicillin and its derivatives as well as to other antibiotics.
  • OrfX has no defined function in S. aureus and is unique to SA.
  • the integration of SCC mec creates a signature unique to MRSA.
  • SCC mec elements have two essential components, the ccr gene complex ( ccr ) and the mec gene complex (mec).
  • the ccr gene complex is composed of ccr genes and surrounding open reading frames (ORFs), and the mec gene complex is composed of the mecA gene, regulatory genes, and insertion sequences upstream or downstream of mecA.
  • ORFs open reading frames
  • mec gene complex is composed of the mecA gene, regulatory genes, and insertion sequences upstream or downstream of mecA.
  • RE types describe the sequence variations of the right extremity junction of the SCC mec transposon.
  • RE2 constitutes the majority of all RE types (representing SCC mec types I, II, III (some) and some IV).
  • RE3 and RE7 represent SCC mec types III and V and SCC mec type V, respectively.
  • the set of primers may comprise
  • oligomeric compound primarily to “oligonucleotides” but also to “modified oligonucleotides” that are able to “prime” DNA synthesis by a template-dependent DNA polymerase, i.e. the 3'-end of the e.g. oligonucleotide provides a free 3'-ooh group whereto further "nucleotides” may be attached by a template-dependent DNA polymerase establishing 3' to 5' phosphodiester linkage whereby desoxynucleoside triphosphates are used and whereby pyrophosphate is released. Therefore, there is - except for the intended function - no fundamental difference between a "primer", an "oligonucleotide” or a “probe” according to the invention.
  • the set of primers consists of a primer specific for MRSA type RE2, a primer specific for MRSA type RE3 and a primer specific for MRSA type RE7, wherein the primers for MRSA type RE2, for MRSA type RE3 and for MRSA type RE7 consist of the sequences of SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94, respectively.
  • primers for MRSA type RE2, for MRSA type RE3 and for MRSA type RE7 comprise or consist of the nucleic acid sequences of SEQ ID NO: 11, SEQ ID NO: 25, and SEQ ID NO: 30, respectively, or wherein the primers for MRSA type RE2, for MRSA type RE3 and for MRSA type RE7 comprise or consist of the nucleic acid sequences of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 32, respectively.
  • the set of primers additionally comprises a further primer specific for methicillin-resistant S . aureus (MRSA) as well as methicillin-sensitive S . aureus (MSSA) (primer for MRSA/MSSA).
  • MRSA methicillin-resistant S . aureus
  • MSSA methicillin-sensitive S . aureus
  • examples of such a primer are selected from the group of primers comprising or consisting of the nucleic acid sequence selected from the group consisting of
  • the number of primer used in the method may be restricted. Accordingly, in a further preferred embodiment of the invention the set of primers consists of at most 5, preferably at most 4 primers. Examples of such sets disclosed herein are :
  • the primer for RE2, RE3, RE7 and/or MRSA/MSSA comprises or consists of a functionally active variant of any of the primers of SEQ ID NO: 1 to 44 or 92 to 95, optionally encompassed in any of the preferred combinations of particular primers as detailed above.
  • a functionally active variant of any of the primers of SEQ ID NO: I to 44 or 92 to 95 may be identified by using the primer in the method of the disclosure.
  • a functionally active variant of a primer of any of the SEQ ID NO: 1 to 44 or 92 to 95 pertains to a primer which provides a similar or higher specificity and sensitivity in the method or kit of the disclosure as compared to the respective sequence of SEQ ID NO: 1 to 44 or 92 to 95.
  • a specificity of 100 % means that the test recognizes all MRSA positive samples as MRSA positive.
  • a similar specificity in the context of the present invention relates to a specificity which is at most 10 %, preferably at most 5 %, more preferably at most 4, 3, 2 or 1 % smaller than that of the respective primer, if the primers (the variant and respective primer without mutation) are tested in the method of the invention, preferably the method as described in Example 3.
  • a sensitivity of 100 % means that the test recognizes all sick people as such. We can always construct a trivial test that achieves 100 % sensitivity by classifying all test cases positive.
  • a similar sensitivity in the context of the present invention relates to a sensitivity which is at most 10 %, preferably at most 5 %, more preferably at most 4, 3, 2 or 1 % smaller than that of the respective primer, if the primers (the variant and respective primer without mutation) are tested in the method of the invention, preferably the method as described in Example 3.
  • the primer comprises or consists of a functionally active variant of any of the primers of SEQ ID NO: 1 to 44 or 92 to 95, wherein
  • the part of the primer may be obtained by terminal deletion of nucleotides (at the 5' end and/or the 3' end). Sequence identity may be determined by sequence alignment. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms have been described e.g. in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 or Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448, 1988 .
  • NCBI Basic Local Alignment Search Tool (BLAST) ( Altschul et al., J. Mol. Biol. 215: 403-410, 1990 ) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Variants of a primer of any of the sequences of SEQ ID NO: 1 to 44 or 92 to 95 are typically characterized using the NCBI Blast 2.x.
  • the variant may e.g. vary from the sequence of SEQ ID NO: 1 to 44 or 92 to 95 by one or more nucleotide additions, deletions or substitutions, particularly one or more nucleotide additions, deletions or substitutions at the 5' end and/or the 3' end of the respective sequence of SEQ ID NO: 1 to 44 or 92 to 95.
  • the primer (and/or the probe) may be chemically modified, i.e. the primer and/ or the probe comprise a modified nucleotide or a non-nucleotide compound.
  • the probe (or the primer) is then a modified oligonucleotide.
  • Modified nucleotides (or “nucleotide analogs") differ from a natural "nucleotide” by some modification but still consist of a base, a pentofuranosyl sugar, a phosphate portion, base-like, pentofuranosyl sugar-like and phosphate-like portion or combinations thereof.
  • a "label” may be attached to the base portion of a “nucleotide” whereby a "modified nucleotide” is obtained.
  • a natural base in a “nucleotide” may also be replaced by e.g. a 7-desazapurine whereby a "modified nucleotide” is obtained as well.
  • the terms "modified nucleotide” or “nucleotide analog” are used interchangeably in the present application.
  • a “modified nucleoside” (or “nucleoside analog") differs from a natural nucleoside by some modification in the manner as outlined above for a "modified nucleotide” (or a “nucleotide analog”).
  • modified oligonucleotide belongs to another specific subgroup of the “oligomeric compounds", that possesses one or more “nucleotides", one or more “non-nucleotide compounds” or “modified nucleotides” as “monomeric units".
  • modified oligonucleotide refers to structures that function in a manner substantially similar to “oligonucleotides” and are used interchangeably throughout the application.
  • a "modified oligonucleotide” (or a “oligonucleotide analog”) can be for example made by chemical modification of "oligonucleotides” by appropriate modification of the phosphate backbone, ribose unit or the nucleotide bases ( Uhlmann and Peyman, Chemical Reviews 90 (1990) 543 ; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 ).
  • Representative modifications include phosphorothioate, phosphorodithioate, methyl phosphonate, phosphotriester or phosphoramidate inter-nucleoside linkages in place of phosphodiester inter-nucleoside linkages; deaza or aza purines and pyrimidines in place of natural purine and pyrimidine bases, pyrimidine bases having substituent groups at the 5 or 6 position; purine bases having altered substituent groups at the 2, 6 or 8 positions or 7 position as 7-deazapurines; sugars having substituent groups at, for example, their 2' position; or carbocyclic or acyclic sugar analogs.
  • Other modifications are known to those skilled in the art.
  • modified oligonucleotides are best described as being functionally interchangeable with, yet structurally different from, natural “oligonucleotides” (or synthetic "oligonucleotides” along natural lines).
  • exemplary modifications are disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO 02/12263 .
  • modification can be made wherein nucleoside units are joined through groups that substitute for the internucleoside phosphate or sugar phosphate linkages. Such linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 .
  • linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 .
  • other than phosphate linkages are utilized to link the nucleoside units, such structures have also been described as "oligonucleosides”.
  • nucleic acid molecule encoding MRSA e.g., nucleic acids encoding alternative portions of portions of RE2, RE3 or RE7
  • OLIGO Molecular Biology Insights Inc., Cascade, CO
  • oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (i.e., the primers need to be long enough to anneal with sequence-specificity and to initiate synthesis but not so long that fidelity is reduced during oligonucleotide synthesis).
  • oligonucleotide primers are 8 to 50 nucleotides in length (e.g. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).
  • sequences of SCC mec of various subtypes of MRSA are available to public, e.g. at the nucleotide data base of NCBI (National Center for Biotechnology Information) (see e.g. AB033763 for type-I staphylococccal cassette chromosome mec : strain NCTC10442; D86934 for type-II staphylococcal cassette chromosome mec : strain N315; AB047089, right extremity of type-III Staphylococcal cassette chromosome mec and its flanking chromosomal region: strain 85/3907; AB063172 for type-IV.1 (IVa) staphylococcal cassette chromosome mec : strain CA05(JCSC1968); or AB121219 type-V staphylococcal cassette chromosome mec : strain JCSC3624(WIS)).
  • NCBI National Center for Biotechnology Information
  • Primer specific for RE2 or “primers specific for RE3” or “primers specific for RE7” as used herein refer to oligonucleotide primers that anneal to nucleic acid sequences encoding RE2 or RE3 or RE7, respectively, and initiate synthesis therefrom under appropriate conditions.
  • the method of the invention uses a pair of probes in order to detect the presence or absence of MRSA.
  • probe refers to synthetically or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies specifically (i.e., preferentially) to "target nucleic acids", in the present case to a MRSA (target) nucleic acid.
  • a “probe” can be identified as a “detection probe” meaning that it detects the target nucleic acid.
  • At least one probe of the pair of probes comprises or consists of a fluorescent moiety and a nucleic acid sequences selected from the group consisting of
  • the first probe comprises or consists of a fluorescent moiety and the nucleic acid sequences of SEQ ID NO: 96 and the second probe comprises or consists of a fluorescent moiety and a nucleic acid sequences of SEQ ID NO: 97 or vice versa.
  • the first MRSA probe of the pair of MRSA probes is labeled with a donor fluorescent moiety and the second MRSA probe of the pair of MRSA probes is labeled with a corresponding acceptor fluorescent moiety. Examples of suitable and preferred labels are listed below, wherein each of the probes may be labeled with any of the labels.
  • a preferred pair of probes is one, wherein the first probe including the first label is fluorescein-5'- AAG TCG CTT TGC CCT TGG GTA -3' (AR mec Fluo 4) and wherein the second probe including the second label is LC-Red 610-5'- ATG CGT TGG TTC GAT TCT TG -3' (AR mec 610 4-MM2).
  • An alternative preferred pair of probes is one, wherein the first probe including the first label is fluorescein-5'- AAG TCG CTT TGC CCT TGG G -3' (AR mec Fluo 4-GV) and wherein the second probe including the second label is LC-Red 610-5'- CAT GCG TTG GTT CGA TTC TTG -3' (AR mec 610 4-MM2-GV).
  • An even more preferred pair of probes is one, wherein the first probe including the first label is 5'- AAG TCG CTT TGC CCT TGG G -3'-fluorescein (MRSA direct Fluos) and wherein the second probe including the second labels is LC-Red 610-5'-CAT GCG TTG GTT CGA TTC TTG -3' (MRSA direct Red 610).
  • the probe comprises or consists of a fluorescent moiety and a functionally active variant of any of the probes of SEQ ID NO: 45 to 76, 96 or 97, particularly wherein
  • a functionally active variant of any of these probes may be identified by using the variant and the respective probe in the method of the invention.
  • a functionally active variant of a probe comprising any of the SEQ ID NO: 45 to 76, 96 or 97 pertains to a probe which has a similar or higher specificity and sensitivity as the respective sequence of SEQ ID NO: 45 to 76, 96 or 97.
  • the terms "specificity” and "sensitivity” are as defined above for the primers and may be determined as detailed for the variants of primers.
  • the part of the probe may be obtained by terminal deletion of nucleotides (at the 5' end and/or the 3' end). Sequence identity may be determined by sequence alignment. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms have been described e.g. in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 or Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448, 1988 .
  • NCBI Basic Local Alignment Search Tool (BLAST) ( Altschul et al., J. Mol. Biol. 215: 403-410, 1990 ) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Variants of a probe comprising any of the sequences of SEQ ID NO: 45 to 76, 96 or 97 are typically characterized using the NCBI Blast 2.x.
  • the variant may e.g. vary from the sequence of SEQ ID NO: 45 to 76, 96 or 97 by one or more nucleotide additions, deletions or substitutions, particularly one or more nucleotide additions, deletions or substitutions at the 5' end and/or the 3' end of the respective sequence of SEQ ID NO: 45 to 76, 96 or 97.
  • the set of primers comprises or consists of
  • Designing oligonucleotides to be used as (hybridization) probes can be performed in a manner similar to the design of primers, although the members of a pair of probes preferably anneal to an amplification product within no more than 5 nucleotides of each other on the same strand such that fluorescent resonance energy transfer (FRET) can occur (e.g., within no more than 1, 2, 3, or 4 nucleotides of each other).
  • FRET fluorescent resonance energy transfer
  • probes can be designed to hybridize to targets that contain a mutation or polymorphism, thereby allowing differential detection of MRSA based on either absolute hybridization of different pairs of probes corresponding to each particular MRSA subtype to be distinguished or differential melting temperatures between, for example, members of a pair of probes and each amplification product generated from a MRSA.
  • oligonucleotide probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are 8 to 50 nucleotides in length (e.g. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).
  • the invention provides methods for detecting the presence or absence of MRSA in a sample. Methods provided by the invention avoid problems of sample contamination, false negatives and false positives.
  • the methods include performing at least one amplifying step and at least one hybridizing step.
  • An amplification step includes contacting the sample with a set of suitable primers to produce an amplification product if a MRSA nucleic acid molecule is present in the sample. Each of the primers anneals to a target within the MRSA target nucleic acid molecule such that at least a portion of the amplification product contains nucleic acid sequence corresponding to the respective MRSA nucleic acid, wherein the set of primers is selected to enable detection of MRSA types I to V.
  • the hybridizing step includes contacting the sample with a pair of MRSA probes.
  • the members of the pair of MRSA probes hybridize to the appropriate amplification product within no more than five nucleotides of each other.
  • a first MRSA probe of the pair of MRSA probes is labeled with a donor fluorescent moiety and a second MRSA probe of the pair of MRSA probes is labeled with a corresponding acceptor fluorescent moiety.
  • the method further includes detecting the presence or absence of FRET between the donor fluorescent moiety of the first MRSA probe and the corresponding acceptor fluorescent moiety of the second MRSA probe. Multiple steps of amplification and hybridisation can be performed, preferably in a thermocycler.
  • amplifying refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid (e.g., MRSA nucleic acid molecules).
  • Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product. The denaturing, annealing and elongating steps each can be performed once.
  • the denaturing, annealing and elongating steps are performed multiple times such that the amount of amplification product is increasing, oftentimes exponentially, although exponential amplification is not required by the present methods.
  • Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g. Taq Polymerase) and an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme (e.g., MgCl 2 and/or KCl).
  • a DNA polymerase enzyme e.g. Taq Polymerase
  • an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme e.g., MgCl 2 and/or KCl.
  • hybridizing refers to the annealing of probes to an amplification product. Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids nonspecific hybridization of the probes.
  • FRET Fluorescence Resonance Energy Transfer
  • Resonance energy transfer is a mechanism by which energy is transferred directly from one molecule to another.
  • the principle of FRET is based on the combined characteristics of the two labels. If a label is excited with a light of a particular wavelength (absorption frequency) its re-emits that energy at a different wavelength (the emission frequency). In FRET the first label is excited with which in turn emits light having the emission frequency. If the emission peak of the first label (donor) overlaps with the excitation peak of the second label (acceptor), proximity of the two labels can be determined, since the first label transfers energy to the second label and the second label emits light at its own emission frequency.
  • the net result is that the donor emits less energy than it normally would (since some of the energy it would radiate as light gets transferred to the acceptor instead), while the acceptor emits more light energy at its excitation frequency (because it is getting extra energy input from the donor fluorophore).
  • FRET fluorescence resonance
  • two oligonucleotide probes each containing a fluorescent moiety, can hybridize to an amplification product at particular positions determined by the complementarity of the oligonucleotide probes to the target nucleic acid sequence, i.e. the MRSA nucleic acid.
  • a FRET signal is generated.
  • the members of the pair of MRSA probes hybridize to the amplification product within no more than five, four or three nucleotides, particularly within no more than two nucleotides of each other, especially within no more than one nucleotide of each other.
  • each probe of the pair of probes is labeled with a suitable label/fluorophore/fluorescent moiety.
  • Labels often referred to as “reporter groups”, “fluorophore” or “fluorescent moiety”, are generally groups that mark or label a target nucleic acid to make it distinguishable from the remainder (nucleic acids having attached a "label” can also be termed labeled nucleic acid binding compounds, labeled probes or just probes).
  • fluorescence resonance energy transfer relationship refers to adjacent hybridization of an "probe” labeled with a “donor fluorescent moiety” and another "probe” labeled with an “acceptor fluorescent moiety” to a "target nucleic acid” such that the "donor fluorescent label” can transfer resonance energy to the "acceptor fluorescent label” such that the "acceptor fluorescent label” produces a measurable fluorescence emission.
  • Suitable labels are known in the art and the skilled practitioner is able to choose a suitable combination of labels for both probes.
  • corresponding refers to an acceptor fluorescent moiety having an emission spectrum that overlaps the excitation spectrum of the donor fluorescent moiety.
  • both signals should be separable from each other.
  • the wavelength maximum of the emission spectrum of the acceptor fluorescent moiety preferably should be at least 30 nm, more preferably at least 50 nm such as at least 80 nm, at least 100 nm or at least 140 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Accordingly, efficient non-radiative energy transfer can be produced therebetween.
  • Fluorescent donor and corresponding acceptor moieties are generally chosen for (a) high efficiency Förster energy transfer; (b) a large final Stokes shift (>100 nm); (c) shift of the emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) shift of the emission to a higher wavelength than the Raman water fluorescent emission produced by excitation at the donor excitation wavelength.
  • a donor fluorescent moiety can be chosen that has its excitation maximum near a laser line (for example, Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, a high quantum yield, and a good overlap of its fluorescent emission with the excitation spectrum of the corresponding acceptor fluorescent moiety.
  • a corresponding acceptor fluorescent moiety can be chosen that has a high extinction coefficient, a high quantum yield, a good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red part of the visible spectrum (> 600 nm).
  • the main target is to obtain signals separable from each other.
  • Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives.
  • acceptor fluorescent moieties depending upon the donor fluorescent moiety used, include LC-Red 610, LC -Red 640, LC-Red 670, LC -Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of Lanthanide ions (e.g., Europium, or Terbium).
  • Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, OR) or Sigma Chemical Co. (St. Louis, MO).
  • Preferred labels according to the invention are fluorescent labels, which are e.g. fluorescent dyes as a fluorescein dye, a rhodamine dye, a cyanine dye, and a coumarin dye.
  • the donor fluorescent moiety may be fluorescein and/or the acceptor fluorescent moiety may be selected from the group consisting of LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, Cy5, and Cy5.5, preferably LC-Red 610 or LC-Red 640. More preferably the donor fluorescent moiety is fluorescein and the acceptor fluorescent moiety is LC-Red 640 or LC-Red 610.
  • the donor-acceptor pair of fluorescein-rhodamine one might use a 470-490 nm excitation filter, and a 500-520 nm emission filter for collecting the light from the fluorescein donor.
  • a 600-650 nm emission filter for collecting the light from the rhodamine acceptor.
  • These emission filters need to collect just the shorter wavelength range of the donor, and just the long emission tail of the acceptor, to avoid cross-talk between the two image channels. That is, one would want to avoid collecting fluorescein emission in the rhodamine channel and vica versa.
  • the problem is that for FRET to work, the donor emission and acceptor excitation spectra must overlap (high overlap is good), but for good signal-to-noise ratio imaging one must avoid collecting the "wrong" photons through a filter.
  • the donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm.
  • the length of each linker arm can be important, as the linker arms will affect the distance between the donor and the acceptor fluorescent moieties.
  • the length of a linker arm for the purpose of the present invention is the distance in ⁇ ngstroms from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 to about 25 ⁇ .
  • the linker arm may be of the kind described in WO 84/03285 .
  • WO 84/03285 also discloses methods for attaching linker arms to particular nucleotide bases, and also for attaching fluorescent moieties to a linker arm.
  • An acceptor fluorescent moiety such as an LC-Red 640(-NHS-ester), LC-Red 610(-NHS-ester) or LC-Red 670(-NHS-ester) can be combined with C6-Phosphoramidites (available from ABI (Foster City, CA) or Glen Research (Sterling, VA)) to produce, for example, LC-Red 640-Phosphoramidite.
  • C6-Phosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)
  • linkers to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, for example, fluorescein-CPG's from Glen Research or ChemGene (Ashland, MA)), amide-linkers (fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG's that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis.
  • FITC-derived for example, fluorescein-CPG's from Glen Research or ChemGene (Ashland, MA)
  • amide-linkers fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex (San Ramon, CA)
  • 3'-amino-CPG's that require coupling of a fluorescein-
  • the detecting step comprises exciting the sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the acceptor fluorescent moiety.
  • the detecting comprises quantitating the FRET.
  • Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system or a fluorometer.
  • Excitation to initiate energy transfer can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range.
  • Hg high intensity mercury
  • Another subject of the invention relates to an alternative method of detecting the presence or absence of methicillin-resistant S . aureus (MRSA) in a sample, the method comprising
  • a common format of FRET technology utilizes two hybridization probes, wherein each probe can be labeled with a different fluorescent moiety and are generally designed to hybridize in close proximity to each other in a target DNA molecule (e.g ., an amplification product) (see above method described first).
  • a target DNA molecule e.g ., an amplification product
  • an alternative FRET format utilizes hydrolyzation probes (second method of the invention) to detect the presence or absence of an amplification product, and hence, the presence or absence of MRSA.
  • This technology utilizes one single-stranded hybridization probe labeled with two fluorescent moieties. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety according to the principles of FRET.
  • the second fluorescent moiety is generally a quencher molecule.
  • the labeled hydrolyzation probe binds to the target DNA (i.e., the amplification product) and is degraded by the 5' to 3' exonuclease activity of the Taq Polymerase during the subsequent elongation phase.
  • the excited fluorescent moiety and the quencher moiety become spatially separated from one another.
  • the fluorescence emission from the first fluorescent moiety can be detected.
  • an ABI PRISM ® 7700 Sequence Detection System uses hydrolyzation probe technology, and is suitable for performing the methods described herein for detecting MRSA.
  • Information on PCR amplification and detection using an ABI PRISM ® 770 system can be found at http://www.appliedbiosystems.com/products.
  • Molecular beacons in conjunction with FRET also can be used to detect the presence of an amplification product using the real-time PCR methods of the invention (alternative second method of the invention).
  • Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety.
  • the second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe.
  • Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g., a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution.
  • the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.
  • Suitable molecular beacons are:
  • fluorescence resonance energy transfer relationship refers to a "probe” labeled with a "donor fluorescent moiety” and an "acceptor fluorescent moiety” such that the “donor fluorescent label” can transfer resonance energy to the "acceptor fluorescent label” such that the "acceptor fluorescent label” produces a measurable fluorescence emission. If the "donor fluorescent label” and “acceptor fluorescent label” are spaced apart by too great a distance, then the “donor fluorescent label” cannot transfer resonance energy to the "acceptor fluorescent label” such that the "acceptor fluorescent label” emits measurable fluorescence, and hence the "donor fluorescent label” and “acceptor fluorescent label” are not in resonance energy transfer relationship.
  • the acceptor fluorescent moiety is a quencher absorbing the energy emitted by the donor fluorescent moiety.
  • the first (using (a) hybridization probe(s)) and second method (using (a) hydrolyzation probe(s) or (a) molecular beacon(s)) according to the invention may be performed in a format for the use in the LightCycler® instrument which is described in US 6,174,670 .
  • This format comprises amplification and detection whereby the latter uses the detection of the fluorescence for the detection of the binding product between a pair of probes or a single probe and the target nucleic acid.
  • These formats apply the FRET technology (see, for example, US Patent Nos. 4,996,143 , 5,565,322 , 5,849,489 , and 6,162,603 ).
  • two probes each containing a fluorescent label, or a single probe containing both fluorescent labels can hybridize to an amplification product at particular positions determined by the complementarity of the probes to the target nucleic acid.
  • the fluorescent label may be a donor or acceptor fluorescent label.
  • a FRET signal is generated.
  • the amplifying step a) in the first method according to the invention comprises contacting the sample with the set of primers and optionally a suitable polymerase, especially by PCR, to produce an amplification product if MRSA nucleic acid is present in said sample
  • said hybridizing step b) comprises contacting said sample with the pair of probes, wherein the members of said pair of probes hybridize to said amplification product within no more than five nucleotides of each other, wherein the first probe of said pair of probes is labeled with a donor fluorescent label and wherein the second probe of said pair of probes is labeled with a corresponding acceptor fluorescent label; and detecting the binding product between the MRSA nucleic acid and the pair of probes in step c) by detecting the presence or absence of FRET between said donor fluorescent label of said first probe and said acceptor fluorescent label of said second probe, wherein the presence of FRET is indicative of the presence of the target nucleic acid in the sample, and wherein the absence of
  • the amplifying step a) in the second method according to the invention comprises contacting the sample with the set of primers and optionally a suitable polymerase, especially by PCR, to produce an amplification product if MRSA nucleic acid is present in said sample, wherein said hybridizing step b) comprises contacting said sample with the probe, wherein the probe is labeled with a donor fluorescent label and a corresponding acceptor fluorescent label, e.g.
  • step c) by detecting the presence or absence of FRET between said donor fluorescent label of the probe and the acceptor fluorescent label, wherein the presence or absence of FRET is indicative of the presence or absence of the target nucleic acid in the sample.
  • PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA).
  • Primers useful in the present invention include oligonucleotides capable of acting as a point of initiation of nucleic acid synthesis within a MRSA nucleic acid sequence.
  • a primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically.
  • a primer is preferably single-stranded for maximum efficiency in amplification.
  • the primer may be produced by chemical synthesis.
  • PCR assays can employ nucleic acid (DNA or RNA) template in general DNA.
  • the template nucleic acid need not be purified; it may be a minor fraction of a complex mixture, such as MRSA toxin nucleic acid contained in human cells.
  • DNA (or RNA) may be extracted from any sample such as body fluids or swabs by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington D.C ).
  • the oligonucleotide primers are combined with other PCR reagents under reaction conditions that induce primer extension.
  • chain extension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.001% (w/v) gelatin, 0.5-1.0 ⁇ g denatured template DNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10 % DMSO.
  • the reactions usually contain 150 to 320 ⁇ M each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof. In certain circumstances, 300 to 640 ⁇ M dUTP can be substituted for dTTP in the reaction.
  • the newly synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction.
  • the steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity amplification products corresponding to the target MRSA nucleic acid molecule.
  • the limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction.
  • the amplification step and the hybridization step are preferably repeated at least once. For use in detection, the number of amplification and hybridization steps will depend, e.g., on the nature of the sample.
  • the sample is a complex mixture of nucleic acids
  • more amplification and hybridization steps may be required to amplify the target sequence sufficient for detection.
  • the amplification and hybridization steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.
  • the presence of the FRET within 55, 45 or 35 cycles of amplification and hybridization is indicative of the presence of MRSA in the sample.
  • the polymerase chain reaction may comprise the steps of adding a thermostable polymerase, nucleotides and primers, whereby a primer is preferably a primer according to the invention, for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the sample with light at a wavelength absorbed by the donor fluorescent label and detecting fluorescent emission from the fluorescence energy transfer pair.
  • thermostable polymerase refers to a polymerase enzyme that is heat stable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand.
  • Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T aqualicus, T. lacteus, T rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR provided the enzyme is replenished.
  • a method for the detection of a MRSA nucleic acid comprises the steps of amplifying the nucleic acid by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the nucleic acid, one of said probes being labeled with an acceptor fluorescent label and the other probe labeled with donor fluorescent label of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target nucleic acid, the donor and acceptor fluorescent labels are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase, nucleotides and primers, whereby the primers are as defined above, for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the sample with light at a wavelength absorbed by the donor label and monitoring temperature dependent fluorescence from the fluorescence energy transfer pair.
  • a method for the detection of a MRSA nucleic acid comprises the steps of amplifying the nucleic acid by polymerase chain reaction in the presence of one nucleic acid probe being labeled with an acceptor fluorescent label, e.g.
  • a quencher and a donor fluorescent label of a fluorescence energy transfer pair, hybridizing the probe with the target nucleic acid
  • said polymerase chain reaction comprising the steps of adding a polymerase optionally having 5' to 3' exonuclease activity, nucleotides, and primers, whereby the primers are as defined above, for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the sample with light at a wavelength absorbed by the donor label and monitoring the fluorescence from the fluorescence energy transfer pair of labels.
  • the nucleic acids can be present in double-stranded or single-stranded form. If the nucleic acid template is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50 %, 60 %, 70 %, 80 %, 90 % or 95 % denatured).
  • predominately denatured e.g., greater than 50 %, 60 %, 70 %, 80 %, 90 % or 95 % denatured.
  • the heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90 °C to about 105 °C for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 0 sec to 4 min.
  • the detecting step is performed after each step of amplification and hybridization and/or in real-time.
  • a detailed description of real-time and on-line monitoring of PCR can be found at http:/Ibiochem.roche.com/lightcycler.
  • the following patent applications describe real-time PCR as used in the LightCycler technology: WO 97/46707 , WO 97/46714 and WO 97/46712 .
  • the LightCycler instrument is a rapid thermal cycler combined with a microvolume fluorometer utilizing high quality optics. This rapid thermocycling technique uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber are controlled by alternating heated and ambient air.
  • the LightCycler® carousel that houses the cuvettes can be removed from the instrument. Therefore, samples can be loaded outside of the instrument (in a PCR Clean Room, for example). In addition, this feature allows for the sample carousel to be easily cleaned and sterilized.
  • the fluorometer as part of the LightCycler® apparatus, houses the light source. The emitted light is filtered and focused by an epi-illumination lens onto the top of the cuvette. Fluorescent light emitted from the sample is then focused by the same lens, passed through a dichroic mirror, filtered appropriately, and focused onto data-collecting photohybrids.
  • the optical unit currently available in the LightCycler® instrument Roche Diagnostics GmBH, Catalog No.
  • 03531414001 includes six band-pass filters (530 nm, Hex excitation, 610 nm, 640 nm, 670 nm, and 705 nm), providing four-color detection and several fluorescence acquisition options. Data collection options include once per cycling step monitoring, fully continuous single-sample acquisition for melting curve analysis, continuous sampling (in which sampling frequency is dependent on sample number) and/or stepwise measurement of all samples after defined temperature interval.
  • the LightCycler® can be operated using a PC workstation and can utilize a Windows operating system, e.g. Windows XP, Windows NT or Windows 2000. Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit.
  • the software can display the fluorescence signals in real-time immediately after each measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence vs. cycle number can be continually updated for all samples. The data generated can be stored for further analysis.
  • the method further comprises determining the melting temperature between one of the two probes and the amplification product of step (a), wherein the melting temperature confirms the presence or the absence of MRSA.
  • Melting curve analysis is an additional step that can be included in a cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA duplexes have separated into single strands.
  • Tm melting temperature
  • the melting temperature of a DNA depends primarily upon its nucleotide composition and its length.
  • DNA molecules rich in G and C nucleotides and/or longer in nucleotides have a higher Tm than those having an abundance of A and T nucleotides and/or shorter in nucleotides.
  • the method further comprises preventing amplification of a contaminant nucleic acid, particularly comprising performing the amplification step (a) in the presence of uracil, especially comprising treating the sample with uracil-DNA glycosylase prior to a first amplifying step.
  • amplification step a) in the presence of uracil
  • an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patent Nos. 5,035,996 , 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.
  • standard laboratory containment practices and procedures are desirable when performing methods of the invention.
  • Containment practices and procedures include, but are not limited to, separate work areas for different steps of a method, containment hoods, barrier filter pipette tips and dedicated air displacement pipettes. Consistent containment practices and procedures by personnel are desirable for accuracy in a diagnostic laboratory handling clinical samples.
  • nucleic acids of the sample Before the nucleic acids of the sample may be analyzed in one of the above-mentioned assays, they might have to be isolated or purified from (biological) samples containing complex mixtures of different components. Often, for the first steps, processes are used which allow the enrichment of the nucleic acids.
  • sample may be used as crude lysate, which may be prepared as follows:
  • the S.E.T.S. kit (Roche Diagnostics Corporation, catalog no. 03753158001) may be use in order to prepare a crude lysate.
  • the method can be exemplarily be described as follows: The sample, e.g. a swab, is broken of the handle and the swab tip is inserted into inner S.E.T.S. tubes (e.g. 0.5 ml tube containing a hole in the bottom and fitting into outer S.E.T.S. tubes, when closed). Tubes are places in outer S.E.T.S. tubes containing silica beads and neutralization buffer (NB). NB neutralizes the transportation medium derived from the swabbing device.
  • inner S.E.T.S. tubes e.g. 0.5 ml tube containing a hole in the bottom and fitting into outer S.E.T.S. tubes, when closed.
  • Tubes are places in outer S.E.T.S. tubes containing silica beads and neutral
  • Adhering material from the swab is removed from the swab tip and transferred into the outer S.E.T.S. tube by gravitation force (centrifugation).
  • the S.E.T.S. tube is closed and heated to 95°C for heat inactivation of bacteria.
  • Cells are disrupted by mechanical force in a shaking device e.g. the MagNALyser® Instrument. Debris is collected in the bottom of the tube by a short spin and the supernatant is used directly as sample in an amplification reaction. The method is described into more detail in Uhl et al., 2005, J Clin Microbiol. 8:4046-51 .
  • a problem encountered during the lysis is that other enzymes degrading the component of interest, e.g. desoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the component of interest during lysis. These degrading enzymes may also be present outside the cells or may have been spatially separated in different cellular compartments before the lysis and come now into contact with the component of interest. Other components released during this process may be e.g. endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy.
  • chaotropic agents as e.g. guanidinium thiocyanate or anionic, cationic, zwitterionic or nonionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases which rapidly degrade these enzymes or unwanted proteins. However, this may produce another problem as the said substances or enzymes can interfere with reagents or components in subsequent steps.
  • Enzymes which can be advantageously used in such lysis or sample preparation processes mentioned-above are enzymes which cleave the amide linkages in protein substrates and which are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3 ).
  • Proteases which have been used in the prior art are e.g. alkaline proteases ( WO 98/04730 ) or acid proteases ( US 5,386,024 ).
  • the protease which is widely used in the prior art for sample preparation for the isolation of nucleic acids is proteinase K from Tritirachium album (see e.g. Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 ) which is active around neutral pH and belongs to a family of proteases known to the person skilled in the art as subtilisins.
  • the component of interest can be further enriched.
  • the non-proteinaceous components of interest are e.g. nucleic acids, they are normally extracted from the complex lysis mixtures before they are used in a probe-based assay.
  • nucleic acids particularly interesting for extraction purposes is the adsorption of nucleic acids to a glass surface although other surfaces are possible.
  • Many procedures for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to glass surfaces. If unmodified nucleic acids are the target, a direct binding of the nucleic acids to a material with a silica surface is preferred because among other reasons the nucleic acids do not have to be modified and even native nucleic acids can be bound.
  • a similar procedure for purifying DNA from lambda phages is described in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201 .
  • the procedure entails the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions and separating the nucleic acids from contaminants such as agarose, proteins or cell residue.
  • the particles may be either centrifuged or fluids are drawn through glass fiber filters. This is a limiting step, however, that prevents the procedure from being used to process large quantities of samples.
  • the use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is more advantageous and described e.g. in Alderton et al., 1992, Anal.
  • the nucleic acids are agglutinated along with the magnetic particles.
  • the agglutinate is separated from the original solvent by applying a magnetic field and performing a wash step. After one wash step, the nucleic acids are dissolved in a Tris buffer.
  • This procedure has a disadvantage, however, in that the precipitation is not selective for nucleic acids. Rather, a variety of solid and dissolved substances are agglutinated as well. As a result, this procedure can not be used to remove significant quantities of any inhibitors of specific enzymatic reactions that may be present.
  • Magnetic, porous glass is also available on the market that contains magnetic particles in a porous, particular glass matrix and is covered with a layer containing streptavidin.
  • This product can be used to isolate biological materials, e.g., proteins or nucleic acids, if they are modified in a complex preparation step so that they bind covalently to biotin.
  • Magnetizable particular adsorbents proved to be very efficient and suitable for automatic sample preparation.
  • Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments are used for this purpose.
  • the most preferred MGPs and methods using magnetic glass particles are those described in WO 01/37291 .
  • the target nucleic acid After the purification or isolation of the nucleic acids including the target nucleic acid from their natural surroundings, the target nucleic acid, the MRSA-specific nucleic acid, may be detected.
  • control samples can be cycled as well.
  • the method of the invention includes a control sample, particularly wherein the control sample comprises MRSA nucleic acid molecule.
  • Control nucleic acid template can be amplified from a positive control (reagent control) sample using, for example, control primers and control probes.
  • Positive control samples can also be used to amplify, for example, a plasmid construct containing MRSA nucleic acid molecules.
  • a plasmid control can be amplified internally (e.g., within each biological sample) (internal control) or in separate samples run side-by-side with the patients' samples.
  • thermocycler run also should include a negative control that, for example, lacks MRSA nucleic acid.
  • Such controls are indicators of the success or failure of the amplification, hybridization, and/or FRET reaction. Therefore, control reactions can readily determine, for example, the ability of primers to anneal with sequence-specificity and to initiate elongation, as well as the ability of probes to hybridize with sequence-specificity and for FRET to occur.
  • a preferred control concept comprises a positive control (detecting a known MRSA DNA), a reagent control (detecting a plasmid comprising the MRSA target sequence) and an internal control (detecting a plasmid comprising the MRSA target sequence, wherein the probe binding site, e.g. that for the MRSA sensor probe, has been substituted by another sequence which does not bind the MRSA sensor probe but which is recognised by the internal control sensor probe; alternatively, the binding sites for both probes may be exchanged).
  • the internal control may be part of each of the reactions, i.e. positive control, reagent control negative control and each sample.
  • IC internal control
  • the sample to be analyzed may be any sample.
  • the sample will in general be a biological sample, preferably a sample from a human subject.
  • Representative biological samples that can be used in practicing the methods of the invention include swabs or body fluids such as a sample selected from the group consisting of a swab of an infected wound, a skin swab, a nasal swab, a throat swab, a groin swab, a axilla swab, a swab from site of an invasive device, a swab from site of possible infection, a blood sample, a urine sample and a perineum swab.
  • Biological sample collection and storage methods are known to those of skill in the art. Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (e.g., calcium alginate or aluminum shaft) are all conditions that can affect the success and/or accuracy of the test result.
  • Biological samples can be processed (e.g., by standard nucleic acid extraction methods and/or using commercial kits) to release MRSA nucleic acid, the biological sample is contacted directly with the PCR reaction components and the appropriate oligonucleotides.
  • a further subject of the disclosure relates to a primer, a set of primers, a probe and/or a pair of probes as defined in any of the above embodiment of the invention.
  • kits comprising:
  • the set of primers, probe and/or pair of probes may be as described for the above (preferred) embodiments or suitable for any of the methods of the invention described above.
  • the kit may comprise a package label or package insert having instructions thereon for using the set of MRSA primers and the pair of MRSA probes to detect the presence or absence of in a sample.
  • Further optional components of the kit of the invention are at least one suitable enzyme, particularly uracil-DNA-glycosylase and/or a DNA polymerase, and/or a suitable buffer.
  • the kit may also contain a template-dependent polymerase having 3' to 5' exonucleolytic activity, preferably the Taq Polymerase, nucleotides and oligonucleotides.
  • a kit is provided comprising a template dependent DNA polymerase, nucleotides and an oligonucleotide or a pair of primers according to the invention.
  • kits known in the art further comprise plastics ware which can be used during the amplification procedure as e.g. microtitre plates in the 96 or 384 well format or just ordinary reaction tubes manufactured e.g. by Eppendorf, Hamburg, Germany and all other reagents for carrying out the method according to the invention.
  • plastics ware which can be used during the amplification procedure as e.g. microtitre plates in the 96 or 384 well format or just ordinary reaction tubes manufactured e.g. by Eppendorf, Hamburg, Germany and all other reagents for carrying out the method according to the invention.
  • the kit contains further reagents for isolating the nucleic acid. Therefore, the kit can additionally contain a material with an affinity to nucleic acids, preferably the material with an affinity to nucleic acids comprises a material with a silica surface. Preferably, the material with a silica surface is a glass. Most preferably, the material with an affinity to nucleic acids is a composition comprising magnetic glass particles as described in WO 96/41811 or WO 01/37291 .
  • the kit can further or additionally comprise a lysis buffer containing e.g. chaotropic agents, detergents or alcohols or mixtures thereof which allows the lysis of cells and separately a protease, e.g.
  • the kit may further or additionally comprise a washing solution which is suitable for the washing step of the magnetic glass particles when DNA or RNA is bound thereto.
  • This washing solution may contain ethanol and/ or chaotropic agents in a buffered solution or solutions with an acidic pH without ethanol and/ or chaotropic agents as described above. Often the washing solution or other solutions are provided as stock solutions which have to be diluted before use.
  • the kit may further or additionally comprise an eluent or elution buffer, i.e. a solution or a buffer (e.g.
  • RNA DNA or RNA bound to the magnetic glass particles.
  • additional reagents or buffered solutions may be present which can be used for the purification process of a nucleic acid, i.e. DNA or RNA.
  • the kit contains inner, outer S.E.T.S. tubes and/or the neutralization buffer as sample prep reagents.
  • the method of the invention is carried out without purification of the samples by preparing crude lysates, e.g. as detailed above.
  • the kit may be adapted to this method in that means for further isolation or purification of nucleic acids are not included into the kit.
  • a preferred embodiment of the present invention is to use the method or the kit of the present invention in automatable methods as e.g. described in WO 99/16781 .
  • Automatable method means that the steps of the method are suitable to be carried out with an apparatus or machine capable of operating with little or no external control or influence by a human being.
  • Automatized method means that the steps of the automatable method are carried out with an apparatus or machine capable of operating with little or no external control or influence by a human being. Only the preparation steps for the method may have to be done by hand, e.g. the storage containers have to filled up and put into place, the choice of the samples has to be done by a human being and further steps known to the expert in the field, e.g. the operation of the controlling computer.
  • the apparatus or machine may e.g. add automatically liquids, mix the samples or carry out incubation steps at specific temperatures.
  • a machine or apparatus is a robot controlled by a computer which carries out a program in which the single steps and commands are specified.
  • Preferred automatized methods are those which are carried out in a high-throughput format which means that the methods and the used machine or apparatus are optimized for a high-throughput of samples in a short time.
  • the methods or the kits according to the present invention are used in semi-automatized process which means that some reaction steps may have to be done manually.
  • a suspension containing MGPs according to the present invention is taken from a storage container and partial volumes are added to different reaction vessels.
  • Reaction vessels may be reaction tubes made from plastics eventually in microtitre plate format contain 96 or 384 or more wells where a reaction can be carried out. However, these vessels may be made from other material e.g. from steel.
  • the kit according to the invention is used for research, bioanalytics or diagnostics.
  • the kit according to the invention or the method according to the invention is used in a high-throughput format, i.e. in an automatized method which allows the analysis of a high number of different samples in a very short time.
  • the LightCycler® MRSA direct test is a qualitative in vitro diagnostic test for the direct detection of methicillin resistant Staphylococcus aureus (MRSA) to aid in the prevention and control of MRSA colonization in e.g. healthcare settings.
  • MRSA methicillin resistant Staphylococcus aureus
  • PCR polymerase chain reaction
  • the assay consisted of the sample prep reagents and amplification reagents. Sample preparation was carried out by extraction of bacteria from swabs and a subsequent lysis step. Lysis can be done either enzymatically or mechanically. For SA mechanical lysis showed good results in the past. In addition mechanical lysis can be done irrespective of the bacteria or bacterial phenotype present in the sample. Therefore, mechanical lysis was the favored method. For further processing of the lysate two options are possible.
  • the assay used amplification by PCR and detection using FRET probes. Controls for amplification and internal controls were included, in order to control amplification and hybridization. Detection was either by amplification curve or melting curve analysis.
  • the primer and probes were designed so that the reverse primer and the pair of probes (detection probes) are located in the S . aureus portion of the target region. Therefore, this element remained the same for all RE types.
  • RE types describe the sequence variations of the right extremity junction of the SCC mec transposon.
  • the RE2 amplicon representing the majority of all RE types has been evaluated by sequencing 93 unrelated specimens ( Fig. 2 ).
  • For this predominant RE type (representing SCC mec type I, II, III (some) and some IV) primers and probes have been designed (see table 1). This setup represents the core assay. Further primers in order to cover SCC types I to V were added when appropriate. (see table 1).
  • RE2 and RE7 single tests using the same probes and the same reverse primer but different forward primers were established (see table 1). These single tests were analyzed for the dynamic range from about 10 6 genomic copies/PCR to 10 copies/PCR in tenfold dilutions ( Figures 3 to 5 ). For data analysis the qualitative detection module and manual Tm calling were employed.
  • a PCR assay for methicillin-resistant Staphylococcus aureus (MRSA) present on nasal swab specimens was developed with primers for amplification and FRET hybridization probes for detection of the amplified target nucleic acid with the LightCycler instrument.
  • the assay was performed as described in the above Examples 1 to 3 unless otherwise noted.
  • Nasal swabs delivered to the laboratory are broken off into a screw capped lysis tube containing 600 ⁇ l of neutralization buffer and approximately 50 ⁇ l of 0.1 mm glass beads.
  • the tube is capped, heated for 2 minutes in a heating block set at 95 to 100C and processed on a MagNA Lyser for 70 seconds at a speed setting of 5000.
  • the tube is centrifuged at 20,000 x g for 1 minute and 5 ⁇ l of the liquid above the swab is used for PCR of MRSA.
  • MRSA Three types of MRSA (RE2, RE3 and RE7) are detected with the PCR assay. Melting curve analysis of the 640 nm signal is used to detect MRSA. All three types of MRSA provide the same melting curve melting temperature. Melting curves above baseline with a T m within +/- 2 °C of the positive control are considered positive. An internal control (IC) template is included in the assay using hybridization probe detection at 710 nm. Detection of the positive control in negative specimens is necessary to demonstrate lack of PCR inhibition.
  • IC internal control
  • Coagulase negative staphylococcus may have a portion of the PCR target. To confirm that isolates of these bacteria do not cross-react in the PCR, DNA from lysed cultures of 29 mecA-negative and 75 mecA-positive coagulase-negative staphylococci were tested. None of the coagulase-negative staphylococci gave a positive result.
  • PCR inhibition was tested by adding various amounts of whole blood to a swab and processing according to the workflow. Spiking with 5 or 10 ⁇ l of whole blood showed no inhibition of the internal control. Adding 20 ⁇ l of blood gave less than 10 % inhibition from replicate tests. Adding 70 ⁇ l of whole blood to the swab produced inhibition of PCR of the internal control with most replicate tests.
  • the sensitivity of the PCR for the RE2, RE3 and RE7 MRSA targets was evaluated by testing in replicates of 5 the number of positive PCR results for dilutions of DNA from each MRSA type.
  • MRSA type DNA copies RE2 RE3 RE7 20 5 5 5 10 5 4 5 5 3 4 3 2.5 0 1 1
  • the sensitivity was ⁇ 90% for all three MRSA types when 10 copies of DNA were used for PCR.

Claims (15)

  1. Verfahren zum Nachweisen der Gegenwart oder Abwesenheit von Methicillinresistentem S. aureus (MRSA) in einer Probe, wobei das Verfahren umfasst
    (a) Durchführen eines Amplifizierungsschrittes umfassend das Inkontaktbringen der Probe mit einem Satz von MRSA-Primern, um ein Amplifikationsprodukt herzustellen, wenn MRSA in der Probe vorhanden ist,
    (b) Durchführen eines Hybridisierungsschritts umfassend das Inkontaktbringen des Amplifikationsprodukts aus Schritt (a) mit einem MRSA-Sondenpaar, wobei die erste MRSA-Sonde des MRSA-Sondenpaars mit einer Donorfluoreszenzkomponente markiert ist und wobei eine zweite MRSA-Sonde des MRSA-Sondenpaars mit einer entsprechenden Akzeptorfluoreszenzkomponente markiert ist; und
    (c) Nachweisen der Gegenwart oder Abwesenheit von Fluoreszenzresonanz-Energietransfer (FRET) zwischen der Donorfluoreszenzkomponente der ersten MRSA-Sonde und der Akzeptorfluoresenzkomponente der zweiten MRSA-Sonde, wobei die Gegenwart von FRET für die Gegenwart von MRSA in der Probe indikativ ist und wobei die Abwesenheit von FRET für die Abwesenheit von MRSA in der Probe indikativ ist,
    wobei das Verfahren zum Nachweisen jeder der Staphylococcus-Chromosomalkassetten (Staphylococcal Chromosomal Cassettes; SCCmec) der Typen I bis V von MRSA in der Lage ist, und
    wobei der Primersatz umfasst
    (i) einen für MRSA Typ RE2 spezifischen Primer, nämlich einen Primer bestehend aus der Nukleinsäuresequenz 5'- TGA AAT GAA AGA CTG CGG AG -3' (MRSA direct RE2 fwd; SEQ ID NO: 92);
    und
    (ii) einen für MRSA Typ RE3 spezifischen Primer, nämlich einen Primer bestehend aus der Nukleinsäuresequenz 5'- CCA CAT CTC ATT AAA TTT TTA AAT TAT ACA C -3' (MRSA direct RE3 fwd; SEQ ID NO: 93);
    und
    (iii) einen für MRSA Typ RE7 spezifischen Primer, nämlich einen Primer bestehend aus der Nukleinsäuresequenz 5'- CAA TCC TTT TTA TAT TTA AAA TAT ATT ATA CAC -3` (MRSA direct RE7 fwd; SEQ ID NO: 94).
  2. Verfahren nach Anspruch 1, wobei der Primersatz zusätzlich einen weiteren Primer enthält, der sowohl für Methicillin-resistenten S. aureus (MRSA) wie auch Methicillin-sensitiven S. aureus (MSSA) spezifisch ist.
  3. Verfahren nach Anspruch 2, wobei der zusätzliche Primer die Nukleinsäuresequenz 5'- CAA GGA AAG ATG CTA TCT TCC G -3' (MRSA direct rev; SEQ ID NO: 95) umfasst oder daraus besteht.
  4. Verfahren nach einem beliebigen der Ansprüche 1 bis 3,
    - wobei der Primersatz aus höchstens 4 oder 5 Primern besteht und/oder
    - wobei der Primersatz MRSA direct RE2 fwd, MRSA direct RE3 fwd und MRSA direct RE7 fwd und einen reversen Primer umfasst oder daraus besteht.
  5. Verfahren nach einem beliebigen der Ansprüche 1 bis 4, wobei der reverse Primer MRSA direct rev ist.
  6. Verfahren nach einem beliebigen der Ansprüche 1 bis 5, wobei mindestens eine Sonde des Sondenpaars eine Fluoreszenzkomponente und eine Nukleinsäuresequenz ausgewählt aus der Gruppe bestehend aus
    - 5'- AAG TCG CTT TGC CCT TGG G -3' (MRSA direct Fluos w/o label; SEQ ID NO: 96), und
    - 5'- CAT GCG TTG GTT CGA TTC TTG -3' (MRSA direct Red 610 w/o label; SEQ ID NO: 97)
    enthält oder daraus besteht.
  7. Verfahren nach einem beliebigen der Ansprüche 1 bis 6, wobei die erste Probe einschließlich des ersten Markers 5'- AAG TCG CTT TGC CCT TGG G -3'-Fluorescein (MRSA direct Fluos) ist, und wobei die zweite Sonde einschließlich des zweiten Labels LC-Red 610 -5' - CAT GCG TTG GTT CGA TTC TTG -3' (MRSA direct Red 610) ist.
  8. Verfahren nach einem beliebigen der Ansprüche 1 bis 7,
    - wobei die Mitglieder des MRSA-Sondenpaars innerhalb von höchstens fiinf, vier oder drei Nukleotiden an das Amplifikationsprodukt hybridisieren;
    und/oder
    - wobei
    (a) die Donorfluoreszenzkomponente Fluoreszein ist; und/oder
    (b) die Akzeptorfluoreszenzkomponente ausgewählt ist aus der Gruppe bestehend aus LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, Cy5 und Cy5.5 ist.
  9. Verfahren zum Nachweisen der Gegenwart oder Abwesenheit von Methicillinresistentem S. aureus (MRSA) in einer Probe, wobei das Verfahren umfasst
    (a) Durchführen eines Amplifizierungsschrittes umfassend das Inkontaktbringen der Probe mit einem Satz von MRSA-Primern, um ein Amplifikationsprodukt herzustellen, wenn MRSA in der Probe vorhanden ist,
    (b) Durchführen eines Hybridisierungsschritts umfassend das Inkontaktbringen des Amplifikationsprodukts aus Schritt (a) mit einer MRSA-Sonde, wobei die MRSA-Sonde mit einer Donorfluoreszenzkomponente und mit einer entsprechenden Akzeptorfluoreszenzkomponente markiert ist; und
    (c) Nachweisen der Gegenwart oder Abwesenheit von Fluoreszenzresonanz-Energietransfer (FRET) zwischen der Donorfluoreszenzkomponente und der Akzeptorfluoreszenzkomponente der MRSA-Sonde, wobei die Gegenwart oder Abwesenheit von FRET indikativ für die Gegenwart bzw. Abwesenheit von MRSA in der Sonde ist,
    wobei das Verfahren zum Nachweisen jeder der Staphylococcus-Chromosomalkassetten (Staphylococcal Chromosomal Cassettes; SCCmec) der Typen I bis V von MRSA in der Lage ist, und wobei der Primersatz wie in den Ansprüchen 1 bis 6 definiert ist.
  10. Verfahren nach Anspruch 9,
    a) wobei die Sonde zwei Fluoreszenzkomponenten umfasst oder daraus besteht,
    b) wobei die Sonde zwei Fluoreszenzkomponenten umfasst oder daraus besteht, wobei die Donorfluoreszenzkomponente Fluoreszein ist; und/oder wobei die Akzeptorfluoreszenzkomponente ausgewählt ist aus der Gruppe bestehend aus LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, Cy5 und Cy5.5;
    c) wobei der Amplifizierungsschritt ein Polymeraseenzym mit 5' nach 3' Exonukleaseaktivität einsetzt;
    d) wobei die Donor- und Akzeptorfluoreszenzkomponenten innerhalb von höchstens 5 Nukleotiden voneinander auf der Sonde sind;
    e) wobei die Sonde eine Nukleinsäuresequenz umfasst, die die Ausbildung einer Sekundärformation erlaubt, wobei die Ausbildung der Sekundärformation in der räumlichen Nähe der Donor- und Akzeptorfluoreszenzkomponente resultiert und/oder
    f) wobei die Akzeptorfluoreszenzkomponente ein Quencher ist.
  11. Verfahren nach einem beliebigen der Ansprüche 1 bis 10,
    a) wobei der Nachweisschritt das Anregen der Probe mit einer Wellenlänge, die von der Donorfluoreszenzkomponente absorbiert wird, und das Sichtbarmachen und/oder Messen der von der Akzeptorfluoreszenzkomponente emittierten Wellenläge umfasst;
    b) wobei das Nachweisen das Quantifizieren von FRET umfasst;
    c) wobei die Gegenwart von FRET innerhalb von 55, 45 oder 35 Zyklen indikativ für die Gegenwart von MRSA in der Probe ist;
    d) wobei der Nachweisschritt nach jedem Amplifizierungs- und Hybridisierungsschritt und/oder in Echtzeit durchgeführt wird;
    e) weiterhin umfassend das Bestimmen der Schmelztemperatur zwischen einer oder beiden Sonde(n) und dem Amplifikationsprodukt von Schritt (a), wobei die Schmelztemperatur die Gegenwart oder Abwesenheit von MRSA bestätigt;
    f) weiterhin umfassend: Verhindern der Amplifikation einer kontaminierenden Nukleinsäure;
    g) weiterhin umfassend: Verhindern der Amplifikation einer kontaminierenden Nukleinsäure durch Durchführen des Amplifizierungsschrittes (a) in Gegenwart von Uracil und Behandeln der Probe mit Uracil-DNA-Glycosylase vor dem ersten Amplifizierungsschritt;
    h) wobei das Verfahren mit einer Kontrollprobe durchgeführt wird;
    i) wobei die Probe eine biologische Probe ist; und/oder
    j) wobei die Probe ausgewählt ist aus der Gruppe bestehend aus einem Abstrich einer infizierten Wunde, einem Hautabstrich, einem Nasenabstrich, einem Halsabstrich, einem Leistenabstrich, einem Achselabstrich, einem Abstrich einer Stelle eines invasiven Geräts, einem Abstrich einer Stelle einer möglichen Infektion, einer Körperflüssigkeit, einer Blutprobe, einer Urinprobe oder einem Darmabstrich.
  12. Zusammensetzung, umfassend einen wie in einem beliebigen der Ansprüche 1 bis 5 definierten Primersatz.
  13. Zusammensetzung nach Anspruch 12, weiterhin umfassend eine Probe und/oder ein Probenpaar wie in einem beliebigen der Ansprüche 6 bis 8 oder 12 definiert.
  14. Kit, umfassend
    (a) einen Primersatz wie in einem beliebigen der Ansprüche 1 bis 5 definiert;
    (b) einen Sondensatz wie in Anspruch 6 oder 8 definiert oder eine Probe wie in Anspruch 10 definiert; und
    (c) eine Donorfluoreszenzkomponente und eine entsprechende, die Sonde(n) markierende Fluoreszenzkomponente,
    wobei der Kit zum Nachweisen jeder der Staphylococcus-Chromosomalkassetten (Staphylococcal Chromosomal Cassettes; SCCmec) der Typen I bis V von MRSA in der Lage ist.
  15. Kit nach Anspruch 14,
    (a) geeignet, das Verfahren nach einem beliebigen der Ansprüche 1 bis 11 durchzuführen;
    (b) weiterhin umfassend eine Packungsmarkierung oder einen Beipackzettel mit darauf befindlichen Anweisungen zur Verwendung des MRSA-Primersatzes und des MRSA-Sondenpaars zum Nachweisen der Gegenwart oder Abwesenheit in einer Probe und/oder
    (c) weiterhin umfassend Uracil-DNA-Glycolase und/oder eine DNA-Polymerase und/oder einen geeigneten Puffer.
EP07857150A 2006-12-29 2007-12-28 Verfahren zum nachweis methicillinresistenter s. aureus sowie primer, sonden und kits dafür Active EP2118306B1 (de)

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CA2348042A1 (en) 2001-06-04 2002-12-04 Ann Huletsky Sequences for detection and identification of methicillin-resistant staphylococcus aureus
US11834720B2 (en) 2005-10-11 2023-12-05 Geneohm Sciences, Inc. Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx
JP5503552B2 (ja) 2007-12-21 2014-05-28 バイオメリュー・エスエイ メチシリン耐性黄色ブドウ球菌の検出
JP5550060B2 (ja) * 2008-10-03 2014-07-16 独立行政法人産業技術総合研究所 そうか病病原菌種のｐｃｒ定量用試薬キット
AU2012356749B2 (en) 2011-12-23 2018-08-02 Biomerieux Detection of mecA variant strains of methicillin-resistant Staphylococcus aureus
EP2834375B1 (de) 2012-04-06 2021-05-05 Geneohm Sciences Canada, Inc. Sequenzen zum nachweis und zur identifizierung von methicillinresistentem staphylococcus aureus (mrsa) vom mrej-typ xxi
US9080217B2 (en) * 2012-09-21 2015-07-14 Roche Molecular Systems, Inc. Methods for detection of Clostridium difficile
CN108498806A (zh) * 2018-04-13 2018-09-07 四川大学 一种反义肽核酸-dna四面体载体复合物及其制备方法和应用
KR102264434B1 (ko) * 2018-11-26 2021-06-14 재단법인 바이오나노헬스가드연구단 황색포도상구균-특이 핵산 프로브 및 이의 용도
CN111549151A (zh) * 2020-05-06 2020-08-18 南京实践医学检验有限公司 一种用于检测多种细菌耐药基因的试剂盒

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DE60304481T8 (de) * 2003-11-07 2007-03-08 Federal Rep. of Germany repr.by the Ministry of Health & Soc.Security, the latter repr. by the Pres. of the Robert Koch Inst. Verfahren zur Identifizierung von Methicilin resistente Staphylococcus aureus (MRSA)
CA2606253A1 (en) * 2005-04-21 2006-10-26 Uti Limited Partnership Pcr for mrsa sccmec typing
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